Oxidative stress plays a key role in the pathophysiology of several major cardiovascular diseases, including atherosclerosis, hypertension, heart failure, stroke and diabetes. ROS (reactive oxygen species) affect multiple tissues either directly or through NO depletion. ROS induce cardiovascular dysfunction by modulating cell contraction/dilation, migration, growth/apoptosis and extracellular matrix protein turnover, which contribute to vascular and cardiac remodelling. Of the several sources of ROS within the cardiovascular system, a family of multisubunit NADPH oxidases appears to be a predominant contributor of superoxide anion. Recent findings suggest a significant role of the genetic background in NADPH oxidase regulation. Common genetic polymorphisms within the promoter and exonic sequences of CYBA, the gene that encodes the p22phox subunit of NADPH oxidase, have been characterized in the context of cardiovascular diseases. This review aims to present the current state of research into these polymorphisms in their relationship to cardiovascular diseases.

OXIDATIVE STRESS AND CARDIOVASCULAR DISEASES

Oxidative stress is defined as an imbalanced redox state where pro-oxidants overwhelm antioxidant capacity, resulting in increased availability of ROS (reactive oxygen species) leading to pathophysiological processes and damage to cellular components [1,2]. The major vascular ROS is O2 (superoxide anion), which inactivates NO, thus impairing relaxation [35]. The reaction product between O2 and NO, peroxynitrite (OONO), constitutes a strong oxidant molecule, which is able to oxidize proteins, lipids and nucleic acids, causing cell damage [6]. Dismutation of O2 by SOD (superoxide dismutase) produces H2O2, a more stable ROS which, in turn, is converted into water by catalase and glutathione peroxidase. H2O2 is capable of modulating both contractile and growth-promoting pathways in VSMCs (vascular smooth muscle cells) [7].

There has recently been a high degree of interest in understanding the potential role of ROS in the development of cardiovascular diseases, where studies favour structural and functional alterations such as endothelial dysfunction, vascular wall remodelling, augmented deposition of extracellular matrix proteins and inflammatory processes [815].

THE NADPH OXIDASE SYSTEM

ROS are produced by a wide variety of enzymatic sources, such as enzymes from the mitochondrial transport chain, NADPH oxidase, xanthine oxidase, cyclo-oxygenases, lipoxygenases and uncoupled NOS (NO synthase) (reviewed by Mueller et al. [14]). However, evidence over recent years has suggested that the predominant cellular source of ROS in the context of cardiovascular diseases is the NADPH oxidase family.

The classical NADPH oxidase was first described and characterized in phagocytes (Figure 1), such as neutrophils, and it was originally thought that the enzyme was restricted to leucocytes and used solely for host defence [16]. The membrane-associated proteins consist of a large subunit, gp91phox (where phox is phagocytic oxidase), and a small subunit, p22phox, known as a flavocytochrome b558, which contains the entire electron transport apparatus of the phagocyte NADPH oxidase and thus may act as a physical conduit for electron transport across the membrane. In addition, there are up to three cytosolic subunits (p47phox, p67phox and p40phox) and a low-molecular-mass G-protein (Rac2). In a resting phagocyte, the NADPH oxidase complex is disassembled, with its components segregated into different parts of the cell. Upon stimulation, the cytosolic components of the complex translocate to the phagosome or plasma membrane and assemble with the integral membrane proteins to form a multisubunit enzyme complex that begins to work [17,18]. Studies over the last two decades have indicated that similar NADPH oxidases are present in a wide variety of non-phagocytic cells and tissues (Figure 1) [1921]. In this sense, several homologues of gp91phox (also defined as Nox2) have been identified, which are known as Nox1, Nox3, Nox4 and Nox5 [19]. In addition, there are also homologues of the cytoplasmic components p47phox and p67phox, which are termed NoxO1 and NoxA1 respectively [22]. These two later homologues appear to have a similar tissue distribution to Nox1 and are, therefore, considered to be involved in Nox1 activation. The small GTPase Rac2 is restricted to leucocytes, but Rac1 is expressed ubiquitously and can substitute for Rac2 [22].

Comparative structure of NADPH oxidases

Figure 1
Comparative structure of NADPH oxidases

Nox family members posses a similar structure and enzymatic function, although they differ in their mechanism of activation (reviewed extensively in [21,105]).

Figure 1
Comparative structure of NADPH oxidases

Nox family members posses a similar structure and enzymatic function, although they differ in their mechanism of activation (reviewed extensively in [21,105]).

Nowadays, the central role of NADPH oxidase both in experimental and human cardiovascular diseases, such as metabolic syndrome [23,24], hypertension [2527], diabetes [28,29], left ventricular hypertrophy and heart failure [19,30,31], renal disease [3234], atherosclerosis [3537] and cerebrovascular disease [38], is widely known. It is important to note that in cardiovascular diseases not only the vascular oxidase, but also the phagocytic NADPH oxidase, plays an important role in O2 production, because monocytes and lymphocytes can infiltrate cardiovascular tissues and facilitate structural and functional alterations [3941].

NADPH oxidase is sensitively regulated by a wide range of (patho)physiologically relevant factors, which include (i) humoral factors, (ii) mechanical factors, and (iii) genetic variants [9,42]. Humoral factors related to cardiovascular diseases have been shown to regulate vascular NADPH oxidase expression and activity [40,4345]. Likewise, humoral factors are also implicated in the activation of NADPH oxidase in mononuclear cells [26,40,46]. On the other hand, mechanical forces, including cyclic stretch and laminar and oscillatory shear stress, stimulate vascular NADPH oxidase [47]. In a previous study, it has been suggested that mechanical forces may play a potential regulatory role in the activation of the phagocytic NADPH oxidase [26]. Genetic factors might regulate NADPH-oxidase-driven O2 production. In fact, genetic defects in the genes encoding four of the phox proteins (gp91phox, p22phox, p47phox and p67phox) are known to cause CGD (chronic granulomatous disease), which is a rare inherited disorder of the innate immune system [48]. In spite of NADPH oxidase being involved in the regulation of the vascular wall, it was not been reported whether CGD patients exhibit cardiovascular abnormalities; however, this was probably due to this information not being included in these publications. Another possibility for the absence of cardiovascular abnormalities in CGD patients may be due the compensatory effects of other Nox homologues. Although the incidence of CGD is estimated to be 1 in 200000-250000 individuals, cardiovascular diseases are a substantial public health problem with >35% of the adult population being affected. Therefore studying genetic variants of the NADPH oxidase subunits and their potential consequences in cardiovascular diseases has gained priority.

NADPH OXIDASE IN CARDIOVASCULAR DISEASES: ROLE OF p22phox POLYMORPHISMS

Among the components of NADPH oxidase, the scientific community has shown over the last decade a particular interest in the gene encoding the p22phox protein, an essential subunit for the functionality of the oxidase [4951]. p22phox binding to Nox proteins leads to protein stabilization, and p22phox is thought to interact with Nox1–Nox4 [21]. The underlying concept is that the Nox proteins and the p22phox protein are stable only as a heterodimer, whereas monomers are degraded by the proteosome. In line with this concept, the importance of the p22phox subunit for phagocyte NADPH oxidase was revealed with the identification of p22phox-deficient CGD patients, who did not have detectable Nox2 proteins [52,53]. In vitro studies revealed an essential role of p22phox on Nox1–Nox4 stability. siRNA (small interfering RNA)-mediated p22phox down-regulation leads to decreased function of Nox1–Nox4 [54,55]. Nevertheless, p22phox-deficient CGD patients did not have obvious phenotypic differences from CGD patients with other underlying mutations. This may be due in part to the small number of cases, the young age of the patients and the limited scope of the clinical examination performed in these patients.

Some experimental findings support a pathophysiological role for p22phox gene polymorphisms, some of which are able to influence NADPH oxidase gene expression and activity in the context of cardiovascular diseases [56,57]. For instance, in an animal model of genetic hypertension, both aortic NADPH oxidase activity and p22phox mRNA expression are enhanced in SHR (spontaneously hypertensive rats) compared with normotensive rats [25], and it has been suggested that this up-regulated p22phox mRNA expression might be regulated by genetic variants in the p22phox gene. In accordance with this possibility, the existence of five genetic variants in the p22phox gene promoter in SHR has been reported, which results in an increase in the transcriptional activity of this gene [58].

Human p22phox is encoded by the CYBA gene, located on the long arm of chromosome 16 at position 24. It spans 8.5 kb and is composed of six exons and five introns which encode an open reading frame of approx. 600 bp [52]. A significant number of genetic polymorphisms have been reported within the promoter and exonic sequences of the p22phox gene, some of which are able to influence gene expression and NADPH oxidase activation, leading to significant functional variation between individuals in oxidative stress (Table 1). Moreover, some of these polymorphisms have been associated with diverse cardiovascular diseases, such as hypertension, CAD (coronary artery disease), myocardial infarction, cerebrovascular disease, and diabetic and non-diabetic nephropathy (Table 2).

Table 1
Human CYBA gene polymorphisms in cardiovascular disease
PolymorphismLocationPosition from ATGAllelic change consequencers numberReference
C242T Exon 4 +214 72His→Tyr rs4673 [52
A640G Exon 6 (3′ UTR) +612 Not described rs1049255 [89
C549T Exon 6 +521 174Ala→Val rs1049254 [52
−930A/G Promoter −930 Activation by C/EBP rs9932581 [92
−675A/T Promoter −675 Activation by HIF-1α Not assigned [97
−852C/G Promoter −852 Not described rs16966671 [78,97
−536C/T Promoter −536 Not described rs13306296 [97
PolymorphismLocationPosition from ATGAllelic change consequencers numberReference
C242T Exon 4 +214 72His→Tyr rs4673 [52
A640G Exon 6 (3′ UTR) +612 Not described rs1049255 [89
C549T Exon 6 +521 174Ala→Val rs1049254 [52
−930A/G Promoter −930 Activation by C/EBP rs9932581 [92
−675A/T Promoter −675 Activation by HIF-1α Not assigned [97
−852C/G Promoter −852 Not described rs16966671 [78,97
−536C/T Promoter −536 Not described rs13306296 [97
Table 2
Association of CYBA polymorphisms with cardiovascular risk factors and disease
PolymorphismCardiovascular riskCardiovascular disease
C242T Essential hypertension [81,82CAD [60,85
 Diabetes [74,76,78,79Cerebrovascular disease [69
 Hypercholesterolaemia [84Nephropathy [69
 Smoking [98 
A640G Diabetes [76CAD [61,62
−930A/G Hypertension [92,94,95 
 Diabetes [77 
−675A/T Hypertension [97 
PolymorphismCardiovascular riskCardiovascular disease
C242T Essential hypertension [81,82CAD [60,85
 Diabetes [74,76,78,79Cerebrovascular disease [69
 Hypercholesterolaemia [84Nephropathy [69
 Smoking [98 
A640G Diabetes [76CAD [61,62
−930A/G Hypertension [92,94,95 
 Diabetes [77 
−675A/T Hypertension [97 

CYBA polymorphisms and cardiovascular diseases

C242T polymorphism

In 1990, Dinauer et al. [52] reported the identification of the C242T polymorphism, which is located in exon 4 at position 214 from the ATG codon. The C242T polymorphism encodes a CAC→TAC codon change, thus resulting in a non-conservative substitution of His72 for a tyrosine residue (Table 1), an alteration that may impair the haem-binding site of the p22phox protein. It has been suggested that this replacement leads to a loss of oxidative function and to a decreased production of ROS and oxidative stress in the vasculature [59].

The association of the CYBA C242T polymorphism with atherosclerosis has been extensively studied over the last decade, although the results have been conflicting within Asian and Caucasian populations (Table 3). Inoue et al. [60] found that the T allele conferred protection against atherosclerosis in a Japanese population, whereas, in contrast, other studies did not find any association between the C242T polymorphism and the severity of CAD detected by coronary angiography [6165]. Finally, some studies even showed the opposite effect, with the T allele being significantly associated with the progression of CAD [6668] and cerebrovascular disease [69,70]. Other studies found no association of the C242T polymorphism with peripheral arterial occlusive disease [71]. In addition, some studies have found no differences in coronary epicardial or microvascular responses to acetylcholine or sodium nitroprusiate according to genotypes of this polymorphism [65,72]. However, other studies have shown that carriers of the CC genotype had a significantly blunted endothelium-dependent dilator response, which was independent of other risk factors or atherosclerosis [73]. In the context of diabetes, the findings available are also diverse (Table 3). Thus it appears that the prevalence of the CC genotype is higher in Type 2 [74], but not in Type 1, [75] diabetic patients. Hodgkinson et al. [76] found that the T allele was associated with a susceptibility to diabetic nephropathy in patients with Type 1 diabetes. In another study, this polymorphism was not associated with diabetic nephropathy secondary to Type 2 diabetes mellitus in 612 subjects from a Chinese population [77]. Interestingly, Doi et al. [78] reported a protective effect of the T allele against end-stage renal disease, but only in the non-diabetic group. In agreement with this, Hayaishi-Okano et al. [79] found that Type 2 diabetic patients with the TC/TT genotype had a significantly lower average intima-media thickness than those with the CC genotype, despite the fact that there were no differences in the risk factors. In acute renal failure, biomarkers of oxidative stress and adverse outcomes were associated in patients carrying the T allele [80]. In relation to hypertension, few studies have been performed (Table 3). Raijmakers et al. [81] reported a lack of association between the C242T polymorphism and pre-eclampsia, whereas Moreno et al. [82] recently reported a significant association of the C242T polymorphism with essential hypertension in a Caucasian population. Interestingly, hypertensive patients with the CC genotype had higher plasma levels of von Willebrand factor than those carrying the TT genotype.

Table 3
Association studies of the C242T polymorphism
Association studiesReferences
With cardiovascular disease  
 Atherosclerosis  
  T allele protects from atherosclerosis [60,85,86
  T allele favours progression of atherosclerosis [6668
  No association of the polymorphism with atherosclerosis [6165,71
  T allele association with increased endothelium-dependent  dilator response [73,84
  No association of the polymorphism with the vasodilator  response [72,83
 Cerebrovascular disease  
  T allele favours progression of cerebrovascular disease [69,70
 Diabetes  
  T allele protects from diabetes [74
  No association of the polymorphism with diabetes [75
  T allele favours nephropathy in diabetes [76
  No association of the polymorphism with nephropathy  in diabetes [77
  T allele protects from cardiovascular complications [74
 Hypertension  
  T allele protects from essential hypertension [82
  No association of the polymorphism with pre-eclampsia [81
 Renal disease  
  T allele protects from renal disease [78
  T allele favours progression of renal disease [80
With functional effects  
 T allele associates with reduced NADPH oxidase activity in  
  Control subjects [88
  Hypertensive patients [82
  Atherosclerotic patients [87
Association studiesReferences
With cardiovascular disease  
 Atherosclerosis  
  T allele protects from atherosclerosis [60,85,86
  T allele favours progression of atherosclerosis [6668
  No association of the polymorphism with atherosclerosis [6165,71
  T allele association with increased endothelium-dependent  dilator response [73,84
  No association of the polymorphism with the vasodilator  response [72,83
 Cerebrovascular disease  
  T allele favours progression of cerebrovascular disease [69,70
 Diabetes  
  T allele protects from diabetes [74
  No association of the polymorphism with diabetes [75
  T allele favours nephropathy in diabetes [76
  No association of the polymorphism with nephropathy  in diabetes [77
  T allele protects from cardiovascular complications [74
 Hypertension  
  T allele protects from essential hypertension [82
  No association of the polymorphism with pre-eclampsia [81
 Renal disease  
  T allele protects from renal disease [78
  T allele favours progression of renal disease [80
With functional effects  
 T allele associates with reduced NADPH oxidase activity in  
  Control subjects [88
  Hypertensive patients [82
  Atherosclerotic patients [87

Such conflicting results on whether this polymorphism contributes to cardiovascular disease are likely to exist because multiple long-standing risk factors and cardiovascular burden confound the possible effect of this polymorphism on polygenic diseases such as diabetes and atherosclerosis. For instance, Schneider et al. [83] found no association of the C242T polymorphism with vasodilation in forearm circulation in 90 Caucasian subjects with hypercholesterolaemia. On the other hand, in another study performed in a large Japanese population (2541 subjects with hypercholesterolemia compared with 2707 subjects without hypercholesterolaemia), Shimokata et al. [84] have found that the T allele of the C242T polymorphism was protective against CAD in men with hypercholesterolaemia, whereas this protective effect was less apparent in those without this condition. Other studies have recently obtained consistent results in high-risk populations. Fan et al. [85] have reported a protective effect of the T allele against the development of CAD in a high-risk Finnish Caucasian population. Likewise, Corsetti et al. [86] have found a significant association of C242T with the risk of coronary events in a high-risk subgroup of post-infarction patients defined by inflammation and hypercholesterolaemia. In this study [86], CC individuals had a hazard ratio of 3.14 (95% confidence intervals, 1.34–7.35; P=0.0084) higher than CT/TT subjects for recurrent coronary events.

Several studies performed over recent years suggest that the C242T polymorphism exerts a functional effect on NADPH oxidase activity in physiological and pathophysiological conditions (Table 3). Guzik et al. [87] demonstrated that the 242T allele was associated with decreased vascular NADPH oxidase activity in saphenous veins of CAD patients, independently of other clinical risk factors. Likewise, our group reported recently [82] that NADPH oxidase activity in blood mononuclear cells (lymphocytes and monocytes) from hypertensive patients with the T allele was significantly lower than that of patients carrying the CC genotype. Wyche et al. [88] have shown that the neutrophil NADPH-oxidase-dependent respiratory burst activity in homozygous healthy American individuals with the T allele was significantly lower than that of wild-type carriers and heterozygous healthy individuals. In these studies, enhanced NADPH oxidase activity was not associated with increased p22phox levels in CC individuals, thus suggesting that the polymorphism is in fact decreasing the ability of p22phox to anchor gp91phox and subsequently altering NADPH oxidase activity. Finally, NADPH oxidase activity in blood mononuclear cells from normotensive subjects with the T allele is similar to that of individuals carrying the CC genotype [82]. It has been suggested that in normotension a limiting factor other than p22phox (namely the quantity and/or degree of activation of any of the other NADPH oxidase subunits) is able to regulate NADPH oxidase and masks the C242T functionality in lymphocytes and monocytes [88].

A640G polymorphism

The A640G polymorphism is located in the 3′-untranslated region of CYBA [89], with no amino acid substitution, and may affect transcriptional rate by means of modifying mRNA processing and stability (Table 1). However, there is controversy about the functionality of this polymorphism. For example, Gardemann et al. [61] analysed 2205 Caucasian males, whose coronary anatomy was defined by coronary angiography, and reported that the G allele was more frequent in subjects without coronary disease, which suggested a protective effect exerted by the G allele [61]. Meanwhile, Park et al. [90] found that exercise training in 59 North Americans with high cardiovascular disease risk factors decreased TBARS (thiobarbituric acid reactive substances) by 16%. In this study [90], the A allele carriers had a greater decrease in TBARS than non-carriers at the A640G locus, indicating the A allele as the protector. Nevertheless, other studies have indicated no association of the A640G with cardiovascular diseases. For example, Zafari et al. [62] found no significant frequency distribution of the genotypes among patients without or with angiographically significant coronary disease in a study performed in 216 North American subjects. Inoue et al. [60] reported that the prevalence of the genotypes of the A640G polymorphism did not differ between 201 controls and 201 patients with CAD in a Japanese population. Other studies have also shown a lack of association of the A640G polymorphism with cardiovascular disease [76,91]. Consistent with these studies, O2 production by human neutrophils in 90 healthy North Americans subjects was not altered by the A640G polymorphism [88].

−930A/G polymorphism

The −930A/G polymorphism is located in the promoter region of CYBA at position −930 from the ATG codon [92]. An analysis of the promoter sequence shows that this polymorphism lies on a potential binding site for C/EBP (CCAAT/enhancer-binding protein) transcription factors (Table 1), and it has been speculated that it might modulate CYBA transcriptional activity [93].

The −930A/G polymorphism was reported to be associated with essential hypertension in a Spanish population of 156 subjects [92]. This association with hypertension has now been borne out in a larger population of 623 subjects [92]. In particular, a significant increase in the G allele frequency was found in hypertensive subjects. The frequencies of the GG, GA and AA genotypes were 0.34, 0.43 and 0.23 for the normotensive group and 0.41, 0.47 and 0.12 for the hypertensive group. The functionality of this polymorphism in hypertension was confirmed by Kokubo et al. [94], in a population of 3652 subjects from Japan, who found that the GG (compared with GA+AA) genotype was also associated with hypertension in the male population. In a Brazilian population, this polymorphism was not associated with end-organ damage in hypertensive patients [95]. Likewise, this polymorphism was not associated with diabetic nephropathy secondary to Type 2 diabetes mellitus in 612 subjects from a Chinese population [77].

Although there is some controversy about the association of this polymorphism with cardiovascular diseases, the relevance of the functionality of this polymorphism in hypertension has been highlighted by a study demonstrating that hypertensive subjects with the GG genotype exhibited significantly increased phagocytic p22phox mRNA and protein levels and enhanced NADPH oxidase activity [93]. Interestingly, NO production was lower in GG hypertensive patients than in AA/AG hypertensive patients, thus suggesting the functionality of the −930A/G polymorphism. In contrast, no differences were found in NADPH oxidase expression and activity between genotypes within the normotensive group. In accordance with this, in a healthy population, no functional impact of this polymorphism on neutrophil NADPH-oxidase-dependent O2 production was found [88].

Mutagenesis experiments have demonstrated a functional role of this polymorphism on the CYBA promoter activity [92]. Thus the overexpression of C/EBPδ was able to induce a greater effect on the transcriptional activity of the G compared with the A allelic CYBA promoter constructs in VSMCs, suggesting a potential involvement of C/EBPδ in the NADPH oxidase activation present in those patients carrying the G allele [93]. The relevance of these findings is underlined by a study reporting increased C/EBPδ levels in experimental hypertension [96].

−675A/T polymorphism

This new genetic variation is located in the promoter region of the CYBA gene at position −675 from the ATG codon [97]. The change of the T allele for the A allele appears to be involved in the elimination of a potential target sequence for the binding of the HIF-1α (hypoxia-inducible factor-1α) transcription factor (Table 1).

Our group has reported recently [97] that the −675A/T polymorphism was associated with essential hypertension. The prevalence of the TT genotype was thus higher in hypertensive compared with normotensive subjects. Interestingly, patients carrying the TT genotype had higher blood pressure values and greater carotid intima-media thickness than TA/AA patients within the hypertensive group.

This polymorphism is also associated with NADPH oxidase activation in phagocytic cells. Thus increased NADPH-oxidase-dependent O2 production has been reported in TT subjects compared with TA/AA subjects [97]. Mutagenesis experiments demonstrated a functional role of this polymorphism in the CYBA promoter activity. The overexpression of HIF-1α was therefore able to induce a positive effect on the transcriptional activity of the T but not the A allelic CYBA promoter constructs in VSMCs, thus suggesting a potential involvement of HIF-1α in the NADPH oxidase activation present in those patients carrying the T allele.

Other CYBA polymorphisms

The involvement of other CYBA allelic variants modulating the expression and activity of the p22phox subunit cannot be discounted. Interestingly, two other genetics changes, the −852C/G polymorphism and −536C/T polymorphism (Table 1), have been characterized in the promoter of CYBA, although they were not associated with hypertension in a study performed in a Caucasian population from southern Europe (865 cases and 496 controls) [97]. Likewise, Doi et al. [78] found no association between the −852C/G polymorphism and end-stage renal disease.

In 1990, Dinauer et al. [52] identified another polymorphism (C549T) located in exon 6 at position 521 from the ATG codon (Table 1). The C549T polymorphism encodes a C→T substitution, which predicts the conservative replacement of Ala174 with a valine residue. In a study performed in 35 cases and 28 controls, Krex et al. [70] found no association between this polymorphism and the occurrence of intracranial aneurysms. Further studies will be necessary to analyse the implication of these polymorphisms in other cardiovascular diseases.

Interaction among CYBA polymorphisms and the environment

LD (linkage disequilibrium) analysis suggests that the CYBA polymorphisms are in a low/moderate LD [97]. Overall, the association of the −930A/G polymorphism with other CYBA polymorphisms is almost absent; however, the C242T, −675A/T and A640G polymorphisms appear to be in a moderate LD [61,97]. Thus the relationships among CYBA polymorphisms may add new insights into the genetic studies of CYBA and NADPH-oxidase-mediated O2 production. In this sense, some findings suggest that a synergistic effect between the −930A/G and C242T polymorphisms is able to modulate NADPH oxidase activity (Figure 2) [82]. The concurrence of the −930GG and 242CC genotypes is associated with increased NADPH oxidase activity in hypertensive patients. In an interesting study, Doi et al. [78] have identified a risk haplotype for non-diabetic end-stage renal disease in the CYBA gene. The CC242/AA640 haplotype was associated with end-stage renal disease after adjusting for confounding factors. Likewise, Park et al. [90] found a CYBA haplotype from C242T and A640G polymorphisms that was associated with differential changes in systemic oxidative stress with aerobic exercise training.

Effect of the interaction between −930A/G and C242T polymorphisms on the activation of NADPH oxidase in essential hypertension

Figure 2
Effect of the interaction between −930A/G and C242T polymorphisms on the activation of NADPH oxidase in essential hypertension

Superoxide production was determined by lucigenin chemiluminescence after PMA (2 mg/l) stimulation in phagocytic cells from hypertensive patients. Values are means±S.E.M. *P<0.05 compared with other combinations; †P<0.05 compared with AG+AA/CT+TT combination. Reproduced from M. U. Moreno, G. San José, A. Fortuño, O. Beloqui, J. Díez and G. Zalba, The C242T CYBA polymorphism of NADPH oxidase is associated with essential hypertension, J. Hypertens. 24 (7), pp. 1299–1306, with permission (http://lww.com).

Figure 2
Effect of the interaction between −930A/G and C242T polymorphisms on the activation of NADPH oxidase in essential hypertension

Superoxide production was determined by lucigenin chemiluminescence after PMA (2 mg/l) stimulation in phagocytic cells from hypertensive patients. Values are means±S.E.M. *P<0.05 compared with other combinations; †P<0.05 compared with AG+AA/CT+TT combination. Reproduced from M. U. Moreno, G. San José, A. Fortuño, O. Beloqui, J. Díez and G. Zalba, The C242T CYBA polymorphism of NADPH oxidase is associated with essential hypertension, J. Hypertens. 24 (7), pp. 1299–1306, with permission (http://lww.com).

In a recent study, He et al. [98] found a relevant association of the C242T polymorphism with the risk of coronary heart disease. The authors [98] reported that, compared with non-smokers with a CC genotype, non-smokers with a CT/TT genotype had a decreased risk of heart disease. On the contrary, smokers with a CT/TT genotype had an increased risk compared with smokers with a CC genotype, thus suggesting a potential interaction between smoking and the CYBA C242T polymorphism in relation to heart disease risk.

PROBLEMS AND FUTURE DIRECTIONS

In most cases, association studies have revealed significant associations between CYBA gene polymorphisms and cardiovascular diseases (Table 2), although the results have not been consistent across studies. For example, results of association in admixture populations may actually be due to differences in the frequencies of the alleles in different ethnic groups. In most of studies, association analyses have been performed by adopting insufficiently stringent thresholds. Finally, the enzymatic and structural characterization of the emerging new members of this oxidase family have not been clearly defined.

In addition, it may be time to bear in mind that the synergistic effect of CYBA variants may identify a particular high-oxidative-stress risk subgroup within the healthy population and the population exhibiting cardiovascular risk factors, including hypertension and diabetes. This may therefore help to explain the contradictory results regarding the association of the CYBA polymorphisms with cardiovascular diseases, and points toward the importance of multiple polymorphism assessment in functional and association studies of complex diseases, including hypertension, diabetes and atherosclerosis. We cannot forget that an environmental component (including the most common cardiovascular risk factors) is relevant and involved in the final phenotype of cardiovascular diseases. In this sense, a paradigm shift is taking place in cardiovascular diagnosis and care, driven by the ability to perform routine genetic analysis of individuals.

CYBA haplotypes for C242T and A640G polymorphisms exhibited differential changes in systemic oxidative stress in response to aerobic exercise training, known to be the most effective non-pharmacological intervention to alleviate oxidative stress [90]. Over the last number of years, the importance that genetic variations have in predicting the efficacy of drug therapy, pharmacogenomics, which is refining individualized approaches to care in cardiovascular patients, has been accepted. Interestingly, several genetic variants of NADPH oxidase may modulate the risk of developing anthracycline-induced cardiotoxicity [99]. Cardiovascular therapy, including antihypertensive drugs, statins and thiazolidinediones, in addition to reducing blood pressure, lowering LDL (low-density lipoprotein)-cholesterol and increasing insulin sensitivity respectively, exhibits other pleiotropic properties, including antioxidant effects by reducing NADPH oxidase activity [100104]. Thus the stratification of cardiovascular patients on the basis of individual CYBA risk haplotypes may be useful in developing novel therapeutic approaches.

Abbreviations

     
  • C/EBP

    CCAAT/enhancer-binding protein

  •  
  • CAD

    coronary artery disease

  •  
  • CGD

    chronic granulomatous disease

  •  
  • HIF-1α

    hypoxia-inducible factor-1α

  •  
  • LD

    linkage disequilibrium

  •  
  • O2

    superoxide anion

  •  
  • phox

    phagocytic oxidase

  •  
  • ROS

    reactive oxygen species

  •  
  • SHR

    spontaneously hypertensive rats

  •  
  • TBARS

    thiobarbituric acid reactive substances

  •  
  • VSMC

    vascular smooth muscle cell

This work was supported by an agreement between FIMA (Fundación para la Investigación Medica Aplicada) and UTE (Union Temporal de Empresas) project CIMA (Centro de Investigación Médica Aplicada), FMMA (Fundación Mutua Madrileña de Automovilismo), and by grant 25/2005 from the Department of Health, Government of Navarra, grant SAF2004-07910 from the Ministry of Science and Technology, and grant RECAVA (Red Temática de Enfermedades Cardiovasculares) RD06/0014/0008 from the Ministry of Health.

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